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Chromatographic methods for quantitative analysis of native, denatured, and aggregated basic fibroblast growth factor
V Sluzky1, Z Shahrokh, P Stratton
1Pharmaceutical R & D, Scios Nova Inc., Mountain View, California.
Pharmaceutical Research
|April 1, 1994
Summary
Stability of human recombinant basic fibroblast growth factor (bFGF) was assessed using advanced chromatography techniques. These methods quantify protein denaturation and aggregation in formulations, aiding future stability studies.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Protein Science
Background:
- Human recombinant basic fibroblast growth factor (bFGF) is crucial in various biological processes.
- Ensuring the stability of therapeutic proteins like bFGF in solution formulations is critical for their efficacy.
- Denaturation and aggregation can compromise the biological activity of bFGF.
Purpose of the Study:
- To develop and validate high-performance liquid chromatography (HPLC) methods for assessing the stability of human recombinant bFGF.
- To quantitatively evaluate bFGF denaturation and aggregation in different solution formulations.
- To establish reliable analytical techniques for future bFGF stability investigations.
Main Methods:
- Reversed-phase HPLC (RP-HPLC) was employed to measure total soluble protein, independent of tertiary structure.
- Heparin affinity chromatography (HepTSK) was utilized for quantitative analysis of native bFGF.
- Fluorescence spectroscopy determined the folding state by monitoring tryptophan emission.
- Size exclusion chromatography (SEC) separated native from denatured bFGF based on unfolding kinetics.
Main Results:
- RP-HPLC effectively quantified total soluble protein content.
- HepTSK provided accurate measurements of native bFGF species.
- Fluorescence spectroscopy confirmed changes in protein folding upon denaturation.
- SEC successfully separated native and denatured bFGF, demonstrating the utility of kinetic separation.
- Chromatographic methods showed good sample recovery, unaffected by tertiary structure changes in SEC.
Conclusions:
- Developed HPLC methods provide quantitative measures of physical and chemical changes in bFGF solution formulations.
- These techniques are valuable tools for assessing bFGF stability and guiding formulation development.
- The combined chromatographic and spectroscopic approaches offer a comprehensive strategy for protein stability evaluation.